Human macrophage migration inhibiton factor (MIF) belongs to the group of so-called lymphokines which comprises biologically active, soluble polypeptides that are secreted by lymphocytes and monocytes or macrophages when these are stimulated by antigens, mitogens or the like. Other examples of lymphokines are immune interferon (.gamma.-interferon), interleukin 1 and 2 and macrophage-activating factor (MAF). These lymphokines control the differentiation, activation and proliferation of various cell types of the immune system.
According to the known state of the art, human MIF consists of a group of polypeptides that inhibit the migration ability of macrophages. Human MIF is secreted not only by activated lymphocytes, T- and B-cells, but also by non-lymphoid cells, for example by growing fibroblasts and certain tumour cells. MIF can be clearly differentiated from .gamma.-interferon, macrophage-activating factor (MAF) and other lymphokines.
Human MIF plays a decisive role in the early phase of an inflammation reaction ("delayed type hypersensitivity reaction"). It induces the differentiation of monocytes and quiescent tissue macrophages to mature inflammatory macrophages. Human MIF and related proteins are therefore important markers for inflammatory conditions and may be useful in the therapy of immune regulation diseases and chronic inflammatory diseases.
The isolation and purification of a MIF protein of 8 kD molecular weight from human mononuclear cells stimulated with concanavalin A is described in European Patent Application EP 162 812. This protein is characterized by a N-terminal amino acid sequence (61 amino acids) and its macrophage migration inhibitory activity, further by its immunoreactivity towards selected monoclonal antibodies. Other MIF proteins of 14 kD, 28 kD and 45 kD are described, but poorly characterized. The MIF protein of 14 kD was found to have the same N-terminal amino acid sequence (amino acids 2 to 19) as the 8 kD MIF protein.
According to EP 162 812, human MIF and its proteins are obtained from cell culture supernatants or filtrates of stimulated human cells. This method limits the availability of human MIF due to inherent problems in culturing suitable human cells, the limited availability of fresh human MIF-producing cells, and cumbersome isolation of a single protein.
The fast progress in recombinant DNA technology in recent years provides the general methods for the preparation of polypeptides in large amounts independent of the primary natural sources of such compounds. Identification of a mRNA or a DNA coding for the desired polypeptide is crucial for the success of this approach. If (partial) amino acid sequence information is available, a chemically synthetized nucleic acid probe may lead to the isolation of coding mRNA or DNA from a mixture of mRNA derived from cells producing the desired polypeptides or from a DNA library, respectively. Although many examples for the isolation of a mRNA or DNA coding for a desired polypeptide have so far become known and the general procedure has been described in principle, each new specific problem requires adaption of the technique to the particular case.
Once a complementary or genomic DNA coding for the desired polypeptide is at hand, preparation of suitable expression vectors, transformation of hosts with these vectors, fermentation of transformed hosts and isolation of the expressed polypeptide follows standard procedures. Here again, these procedures must be adapted to the particular problem in order to get stable incorporation of the DNA and sufficiently high expression of the desired polypeptide in a chosen host organism, and acceptable yields of pure, biologically active isolated protein.
Furthermore recombinant DNA technology allows one to produce polypeptide variants by mutating or otherwise altering the coding DNA incorporated in a host organism, thereby enlarging the potential applications of an active principle found in a single polypeptide structure in nature.
A recent publication on cystic fibrosis antigen (CF antigen) isolated from chronic myeloid leukemia cells (J.R. Dorin et al., Nature 1987, 326, 614) suggests that this CF antigen is identical or at least very much related to the MIF-related protein MRP-8 of this invention. However, the present invention provides evidence that MRP-8 is not indicative for cystic fibrosis. At the same time a method of reliable diagnosis of cystic fibrosis is described by this invention based on the immunological determination of another MIF-related protein, MRP-14.